Pathogenic poultry viruses are not only debilitating to chickens, but they also are costly to chicken breeders because most of the resulting diseases are contagious and the poultry industry relies heavily on confined, large-scale breeding facilities. Vaccinating young chicks is often the only viable means to combat these viruses. Although attenuated or killed poultry viral vaccines remain important in the market place, in recent years significant resources have been expended on developing vaccines containing recombinant viral constructs which express pathogenic viral protein antigens. Furthermore, substantial efforts have been made to construct stable and efficacious multivalent recombinant non-pathogenic Marek's Disease virus (rMDVnp) vectors that express foreign genes from multiple viral pathogens. Such multivalent vaccines would serve to minimize the number of injections given to the chicks and thereby, reduce discomfort and stress on the vaccinated chick, as well as significantly reduce costs in labor and materials. Vaccinating with such single multivalent constructs also would be preferable to alternative multivalent rMDVnp vaccines that contain multiple recombinant monovalent rMDVnp constructs, because these alternative vaccines have, at least to date, resulted in protection against only a single viral pathogen. The failure of such alternative vaccines is presumably due to one of the monovalent rMDVnp constructs overgrowing the other monovalent rMDVnp constructs thereby, preventing these other monovalent rMDVnp constructs from inducing a significant immune response. In any case, despite substantial efforts in the past to construct stable and efficacious multivalent recombinant rMDVnp vectors that express foreign genes from multiple viral pathogens heretofore, such efforts have proved unsuccessful.
One poultry virus disease that can be controlled through vaccination is Marek's disease. Marek's disease is a pathogenic disease that adversely affects chickens, worldwide. Marek's disease occurs predominantly in young chickens between 2 and 5 months of age. Clinical signs include: progressive paralysis of one or more of the extremities, incoordination due to paralysis of legs, drooping of the limb due to wing involvement, and a lowered head position due to involvement of the neck muscles. In acute cases, severe depression may result. Bursal and thymic atrophy may also develop.
The etiological agent for Marek's disease is Marek's disease virus serotype 1 (MDV1), a cell-associated virus having a double-stranded DNA genome. MDV1 is a lymphotropic avian alphaherpesvirus that both: (i) infects B cells, which can result in cytolysis, and (ii) latently infects T cells, which can induce T-cell lymphoma. Closely related to the virulent MDV1 strain, Marek's disease virus serotype 2 (MDV2), previously known as Gallid herpes virus 3, is a naturally attenuated MDV strain that has been shown to have little to no pathogenicity in chickens [Petherbridge et al., J. Virological Methods 158:11-17 (2009)]. SB-1 is a specific MDV2 strain that has been shown to be useful in vaccines against MDV1 [see e.g., Murthy and Calnek, Infection and Immunity 26(2) 547-553 (1979)].
Another closely related alphaherpesvirus, Marek's disease virus serotype 3 (MDV3), more widely known as herpesvirus of turkeys (HVT), is a nonpathogenic virus of domestic turkeys [see e.g., Kingham et al., J. of General Virology 82:1123-1135 (2001)]. Two commonly used strains of HVT are the PB1 strain and the FC126 strain. Whereas, HVT is also nonpathogenic in chickens, it does induce a long-lasting protective immune response in chickens against MDV1. Accordingly, HVT has been used in poultry vaccines against virulent MDV1 for many years, generally in combination with SB-1, which is more viraemic than HVT, but considered less safe. Alternatively, when flocks are challenged with particularly virulent MDV1 strains, HVT can be combined with the Rispen's vaccine. The Rispen's vaccine is an isolate that originated from a mildly virulent MDV1 strain that was subsequently further weakened by cell passaging. The Rispen's strain however, retains some virulence towards highly susceptible lines of chickens.
The sequence of the complete genome of HVT has been disclosed [Afonso et al., J. Virology 75(2):971-978 (2001)], and as most alphaherpesviruses, HVT possesses a significant number of potential nonessential insertion sites [see e.g., U.S. Pat. No. 5,187,087; U.S. Pat. No. 5,830,745; U.S. Pat. No. 5,834,305; U.S. Pat. No. 5,853,733; U.S. Pat. No. 5,928,648; U.S. Pat. No. 5,961,982; U.S. Pat. No. 6,121,043; U.S. Pat. No. 6,299,882 B1]. HVT also has been shown to be amenable to genetic modification and thus, has been used as a recombinant vector for many years [WO 87/04463]. Accordingly, recombinant HVT vectors have been reported to express foreign genes that encode antigens from e.g., Newcastle Disease Virus (NDV), [Sondermeijer et al., Vaccine, 11:349-358 (1993); Reddy et al., Vaccine, 14:469-477 (1996)], Infectious Bursal Disease Virus (IBDV), [Darteil et al., Virology, 211:481-490 (1995); Tsukamoto et al., J. of Virology 76(11):5637-5645 (2002)], and Infectious Laryngotracheitis Virus (ILTV) [Johnson et al., Avian Disease, 54(4):1251-1259 (2010); WO 92/03554; U.S. Pat. No. 6,875,856]. The entire genomic sequence of MDV2 is also known [see, GenBank acc. nr: AB049735.1, and Petherbridge et al., supra]. The genomic organization of the MDV2 is very similar to that of HVT, with the US region in particular, being identical to that of HVT [see, Kingham et al., supra].
In addition a recombinant chimeric virus, known as the novel avian herpesvirus (NAHV), has been constructed in which specific regions of the HVT genome have been replaced by the corresponding regions of the MDV1 genome. The NAHV also has been used to express foreign genes that encode antigens from other poultry viruses [U.S. Pat. No. 5,965,138; U.S. Pat. No. 6,913,751].
Like MDV, infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that adversely affects chickens, worldwide [Fuchs et al., Veterinary Research 38:261-279 (2007)]. ILTV causes acute respiratory disease in chickens, which is characterized by respiratory depression, gasping, and expectoration of bloody exudate. Viral replication is limited to cells of the respiratory tract, where in the trachea the infection gives rise to tissue erosion and hemorrhage.
Newcastle disease is another highly contagious and debilitating disease of chickens. The etiological agent for Newcastle disease is the Newcastle disease virus (NDV). NDV belongs to the order of the Mononegavirales and is in the family of Paramyxoviridae. Newcastle disease viruses have a non-segmented, negative sense, single-stranded RNA genome. NDV has been grouped into three distinct pathotypes according to their virulence. Infection of poultry by the non-pathogenic lentogenic strains of NDV is essentially asymptomatic. In direct contrast, the mesogenic (medium pathogenic) and velogenic (highly pathogenic) NDV strains cause extensive disease that can be fatal. Most types of NDV infect the respiratory system and/or the nervous system, and can result in gasping and torticollis.
Infectious bursal disease virus (IBDV), also called Gumboro disease virus, is the causative agent of infectious bursal disease. IBDV causes an acute, highly-contagious, viral infection of a chicken's lymphoid tissue, with its primary target being the bird's essential immunological organ: the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate to high mortality rates. Chicks that recover from the disease may have immune deficiencies because of destruction of (or parts of) the bursa of Fabricius. This makes them particularly vulnerable to secondary infections.
IBDV is a member of the Birnaviridae family. The viruses in this family have a genome consisting of two segments (A and B) of double-stranded RNA. Two serotypes of IBDV exist, serotype 1 and 2, which can be differentiated by virus neutralization (VN) tests. Serotype 1 viruses have been shown to be pathogenic to chickens, while serotype 2 viruses cause only sub-acute disease in turkeys. Historically, IBDV serotype 1 viruses consisted of only one type that is now known as “classic” IBD virus. More recently, so-called “variant” IBDV strains have emerged. Classic and variant strains of IBDV can be identified and distinguished by a virus neutralisation test using a panel of monoclonal antibodies, or by RT-PCR [Wu et al., Avian Diseases, 51:515-526 (2007)]. Well-known classic IBDV strains include, D78, Faragher 52/70, and STC, whereas 89/03 is a well-known variant strain. Many live or inactivated IBDV vaccines are commercially available, e.g. a live vaccine such as NOBILIS® Gumboro D78 (MSD Animal Health).
As indicated above, because HVT can act as both an antigen that provides significant protection against Marek's Disease and as a recombinant vector, it is presently used as a platform vector for such multivalent vaccines as Innovax®-ILT (sold by Merck Animal Health), which protects against ILTV; and Innovax®-ND-SB (sold by Merck Animal Health) and Vectormune® HVT-NDV (sold by Ceva), both of which protect against NDV. Notably, however, heretofore, no multivalent vaccine comprising a recombinant HVT encoding antigens from more than one pathogen has been shown to be stable and efficacious, even though such vaccines had been suggested more than fifteen years ago [see e.g., U.S. Pat. No. 5,965,138]. Indeed, Innovax®-ILT contains the only recombinant HVT that comprises two foreign genes, i.e., ILTV gD and ILTV gI, which has proved to be safe, effective, and stable. However, these two foreign genes are from the same pathogen and moreover, they naturally overlap and need to be co-expressed in order to allow proper immunization against ILTV.
Accordingly, despite the clear advantages of stable, multivalent, recombinant MDVnp constructs that can efficaciously express foreign antigens from two or more different pathogens, and the substantial efforts to design them, heretofore, none have been forthcoming. Therefore, there is a clear need to overcome the collective industry failure, by constructing novel, stable, recombinant MDVnp vectors that can be used in multivalent vaccines as the sole active to protect against two or more different non-MDV1 poultry virus pathogens.
The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.